The aromatic amino acid hydroxylase genes AAH1 and AAH2 in Toxoplasma gondii contribute to transmission in the cat

The Toxoplasma gondii genome contains two aromatic amino acid hydroxylase genes, AAH1 and AAH2 encode proteins that produce L-DOPA, which can serve as a precursor of catecholamine neurotransmitters. It has been suggested that this pathway elevates host dopamine levels thus making infected rodents less fearful of their definitive Felidae hosts. However, L-DOPA is also a structural precursor of melanins, secondary quinones, and dityrosine protein crosslinks, which are produced by many species. For example, dityrosine crosslinks are abundant in the oocyst walls of Eimeria and T. gondii, although their structural role has not been demonstrated, Here, we investigated the biology of AAH knockout parasites in the sexual reproductive cycle within cats. We found that ablation of the AAH genes resulted in reduced infection in the cat, lower oocyst yields, and decreased rates of sporulation. Our findings suggest that the AAH genes play a predominant role during infection in the gut of the definitive feline host

Toxoplasma gondii merozoite gene expression analysis with comparison to the life cycle discloses a unique expression state during enteric development

BACKGROUND: Considerable work has been carried out to understand the biology of tachyzoites and bradyzoites of Toxoplasma gondii in large part due to in vitro culture methods for these stages. However, culturing methods for stages that normally develop in the gut of the definitive felid host, including the merozoite and sexual stages, have not been developed hindering the ability to study a large portion of the parasite’s life cycle. Here, we begin to unravel the molecular aspects of enteric stages by providing new data on merozoite stage gene expression. RESULTS: To profile gene expression differences in enteric stages we harvested merozoites from the intestine of infected cats and hybridized mRNA to the Affymetrix Toxoplasma GeneChip. We analyzed the merozoite data in context of the life cycle by comparing it to previously published data for the oocyst, tachyzoite, and bradyzoite stages. Principal component analysis highlighted the unique profile of merozoites, placing them approximately half-way on a continuum between the tachyzoite/bradyzoite and oocyst samples. Prior studies have shown that antibodies to surface antigen one (SAG1) and many dense granule proteins do not label merozoites: our microarray data confirms that these genes were not expressed at this stage. Also, the expression for many rhoptry and microneme proteins was drastically reduced while the expression for many surface antigens was increased at the merozoite stage. Gene Ontology and KEGG analysis revealed that genes involved in transcription/translation and many metabolic pathways were upregulated at the merozoite stage, highlighting unique growth requirements of this stage. To functionally test these predictions, we demonstrated that an upstream promoter region of a merozoite specific gene was sufficient to control expression in merozoites in vivo. CONCLUSIONS: Merozoites are the first developmental stage in the coccidian cycle that takes place within the gut of the definitive host. The data presented here describe the global gene expression profile of the merozoite stage and the creation of transgenic parasite strains that show stage-specific expression of reporter genes in the cat intestine. These data and reagents will be useful in unlocking how the parasite senses and responds to the felid gut environment to initiate enteric development. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/1471-2164-15-350) contains supplementary material, which is available to authorized users

\u3cem\u3eSarcocystis Neurona\u3c/em\u3e Diagnostic Primer and Its Use in Methods of Equine Protozoal Myeloencephalitis Diagnosis

An amplification primer and probe which can be used in an in vitro diagnostic test for the presence of S. neurona in equine blood or cerebrospinal fluid. Sarcocystis neurona is responsible for the equine condition of protozoal myelitis. The amplification primer is seventeen nucleotides in length and complementary to a unique section of the small ribosomal subunit of Sarcocystis neurona. The primer encompasses nucleotide positions 1470-1487 of the small ribosomal subunit of S. neurona. The primer has the sequence 5\u27 CCATTCCGGACGCGGGT SEQ ID NO:1

Role of Decomposers in Agricultural Waste Management

In this chapter, agricultural waste residue management by bio-organisms is discussed along with different types of decomposition processes. Tons of agricultural wastes are produced every year. These agricultural wastes create major environmental problems without effective means of management methods. There are many technologies being used for the decomposition, which mainly include anaerobic decomposition, compositing, fermentation, etc. All these decomposition processes depend upon the different soil-inhabiting microbes. These microbes are the key components of agri-residue decomposition process. Every step of decomposition requires different microbes. Various sets of catalytical enzymes are involved for the catabolic procedures of organic matter. By successive catabolic reactions, all the organic matters are mineralized into soil essential constituents, which will be the most effective sources of macro- and micronutrients for the soil fertility. Working efficiency of these microbes depends upon different parameters like moisture, temperature, pH, etc. The vitality and efficiency of microbes can be enhanced by using various inert carriers. If the efficiency of these soil microbes enhances by various factors, then the rate of decomposition could be enhanced to handle this ever-increasing problem of agriculture residue in near future

Expanding the Known Repertoire of C-Type Lectin Receptors Binding to Toxoplasma gondii Oocysts Using a Modified High-Resolution Immunofluorescence Assay

The environmental stage of the apicomplexan Toxoplasma gondii oocyst is vital to its life cycle but largely understudied. Because oocysts are excreted only by infected felids, their availability for research is limited. We report the adaptation of an agarose-based method to immobilize minute amounts of oocysts to perform immunofluorescence assays. Agarose embedding allows high-resolution confocal microscopy imaging of antibodies binding to the oocyst surface as well as unprecedented imaging of intracellular sporocyst structures with Maclura pomifera agglutinin after on-slide permeabilization of the immobilized oocysts. To identify new possible molecules binding to the oocyst surface, we used this method to screen a library of C-type lectin receptor (CLR)-human IgG constant region fusion proteins from the group of related CLRs called the Dectin-1 cluster against oocysts. In addition to CLEC7A that was previously reported to decorate T. gondii oocysts, we present experimental evidence for specific binding of three additional CLRs to the surface of this stage. We discuss how these CLRs, known to be expressed on neutrophils, dendritic cells, or macrophages, could be involved in the early immune response by the host, such as oocyst antigen uptake in the intestine. In conclusion, we present a modified immunofluorescence assay technique that allows material-saving immunofluorescence microscopy with T. gondii oocysts in a higher resolution than previously published, which allowed us to describe three additional CLRs binding specifically to the oocyst surface. IMPORTANCE Knowledge of oocyst biology of Toxoplasma gondii is limited, not the least due to its limited availability. We describe a method that permits us to process minute amounts of oocysts for immunofluorescence microscopy without compromising their structural properties. This method allowed us to visualize internal structures of sporocysts by confocal microscopy in unprecedented quality. Moreover, the method can be used as a low- to medium-throughput method to screen for molecules interacting with oocysts, such as antibodies, or compounds causing structural damage to oocysts (i.e., disinfectants). Using this method, we screened a small library of C-type lectin receptors (CLRs) present on certain immune cells and found three CLRs able to decorate the oocyst wall of T. gondii and which were not known before to bind to oocysts. These tools will allow further study into oocyst wall composition and could also provoke experiments regarding immunological recognition of oocysts.Peer Reviewe

Physical Stability and Bio-Efficacy Enhancement of Neem Kernel Aqueous Extract by Optimized Amount of Botanical Synergist for the Control of Early Stages of Mosquitoes

297-303The aim of present study is to enhance the stability of physico-chemical characteristics of neem kernel aqueous extract by botanical stabilizer system. There are variety of bioactive constituents are present in neem which give broad-spectrum of insecticidal activity. Neem aqueous extract is commonly used and found very effective in pest control applications without harming the environment. However, due to hydrolytically unstable characteristics of neem active ingredients which results its lesser bioactivity and limits its usage in aqueous form. To overcome this un-stability issue oil extracted botanical stabilizer (Prosopis juliflora) (Junglee kikar)) were used in various ratios. In 70-30 (NKP-KP) composition (NKP- Neem kernel powder; KP- Kiker powder), neem aqueous extract was found stable without any turbidity, pH change, and fungal growth. Active ingredient, Azadirachtin was found stable with very less degradation i.e only 20–30% degradation. This may possibly be due to inhibition of hydrolytic reactions. Bio-efficacy evaluation data also showed improved and stable mosquito larvae mortality per cent i.e 75–90% with 8 µg/g LD50 value. The approach used in this study could be very useful in long term stability of neem kernel extract in various geographical conditions without adding toxic solvents or chemical compositions

Ultrastructure of Sarcocystis bertrami sarcocysts from a naturally infected donkey (Equus asinus) from Egypt

There is considerable confusion concerning Sarcocystis species in equids. Little is known of Sarcocystis infections in donkeys (Equus asinus). Here we describe the structure of Sarcocystis bertrami-like from the donkey by light microscopy (LM) and transmission electron microscopy (TEM). Nineteen sarcocysts from the tongue of a donkey from Egypt were studied both by LM and TEM. By LM, all sarcocysts had variably shaped and sized projections on the sarcocyst walls, giving it a thin-walled to thick-walled appearance, depending on individual sarcocyst and plane of section. By TEM, sarcocysts walls had villar protrusions (vp) of type 11. The sarcocyst wall had conical to slender vp, up to 6 µm long and 1 µm wide; the vp were folded over the sarcocyst wall. The total thickness of the sarcocyst wall with ground substance layer (gs) was 1-3 µm. The vp had microtubules (mt) that originated deeper in the gs and continued up to the tip. The apical part of the vp had electron dense granules. The mt were configured into 3 types: a tuft of electron dense mt1 extending the entire length of the vp with a tuft of medium electron dense mt2 appearing in parallel, and fine mt3 present only in the villar tips. The gs was mainly smooth with few indistinct granules. All sarcocysts were mature and contained metrocytes and bradyzoites. Bradyzoites were approximately 11-15 × 2-3 µm in size with typical organelles.http://journals.cambridge.org/action/displayJournal?jid=PAR2016-07-30hb201

Seroepidemiology of \u3cem\u3eSarcocystis neurona\u3c/em\u3e and \u3cem\u3eNeospora hughesi\u3c/em\u3e Infections in Domestic Donkeys (\u3cem\u3eEquus asinus\u3c/em\u3e) in Durango, Mexico

There is currently no information regarding Sarcocystis neurona and Neospora hughesi infections in donkeys in Mexico. Here, we determined the presence of antibodies against S. neurona and N. hughesi in donkeys in the northern Mexican state of Durango. Serum samples of 239 domestic donkeys (Equus asinus) were assayed for S. neurona and N. hughesi antibodies using home-made enzyme-linked immunoassays; six (2.5%) of the 239 donkeys tested seropositive for S. neurona. The seroprevalence of S. neurona infection was comparable among donkeys regardless of their origin, health status, or sex. Multivariate analysis showed that seropositivity to S. neurona was associated with increased age (OR = 2.95; 95% CI: 1.11-7.82; p = 0.02). Antibodies to N. hughesi were found in two (0.8%) of the 239 donkeys. Both exposed donkeys were healthy, 3- and 6-year-old females. This is the first evidence of S. neurona and N. hughesi infections in donkeys in Mexico

Antibodies to Toxoplasma gondii and Leishmania spp. in domestic cats from Luanda, Angola

Toxoplasma gondii and Leishmania spp. are zoonotic protozoa of importance to animal and public health. The present study aimed to assess for the first time the seroprevalence of these zoonotic parasites in a domestic feline population living in Luanda, Angola. One hundred and two cats were sampled at a veterinary medical centre, from May 2014 to February 2016. The age of the cats ranged from 2.5 to 143 months (median: 12 months; interquartile range: 7.5–24). Serum samples were tested for immunoglobulin (Ig) G antibodies to T. gondii at two-fold dilutions of 1:20 to 1:2560 with a modified agglutination test (MAT) commercial kit. The direct agglutination test (DAT) for titration of IgG antibodies specific to Leishmania spp. used a standard freeze-dried antigen at a concentration of 5 × 10 7 promastigotes per milliliter, following a predefined protocol. Two-fold dilution series ranging from 1:25 to 1:800 were tested, with a cut-off titre of 100 chosen for seropositivity. Four out of 102 cats (3.9%; 95% confidence interval [CI]: 1.1–9.7) had antibodies to T. gondii: one had a titer of 20, one a titer of 160, and two had a titer ≥ 2560. No cat (0.0%; CI: 0.0–3.5) was found seropositive for Leishmania spp. A statistically significant difference was found between T. gondii seroprevalence and Leishmania spp. seroprevalence (p = 0.043). The odds of a cat being seropositive to T. gondii increased by an average factor of 1.58 for each 1-year increase in age (p = 0.003). The sampled cats were well-cared animals and may not represent the overall feline population of Angola at the national and city levels. The fact that only 12 out of the 102 sampled cats ate or had access to raw or undercooked meat and/or viscera may have reduced the likelihood of finding seropositive results. Under these circumstances, additional studies, including a larger number of cats, are necessary for a more comprehensive assessment of the zoonotic risk posed by these animals in Angola.The authors would like to express their gratitude to Hugo Vilhena for his logistic support. This work was sponsored by the Foundation for Science and Technology (FCT), Ministry of Education and Science, Portugal, under the Projects UID/CVT/00772/2013 and UID/CVT/ 0772/2016.info:eu-repo/semantics/publishedVersio

Occurrence of antibodies to Toxoplasma gondii in scavenging black vultures (Coragyps atratus) in Brazil

Este e o primeiro relato de infecção por Toxoplasma gondii em urubus (Coragyps atratus) que são aves carniceiras obrigatórias, encontradas no continente americano. Amostras de soro de 121 urubus, capturados em área urbana da cidade de São Paulo, Brasil, foram testadas quanto a presença de anticorpos anti-T. gondii pelo teste de aglutinação modificada (MAT, ponto de corte de 1:5). Anticorpos foram encontrados em 16 (13,2%) aves com títulos de 1:5 (6 aves), 1:10 (8 aves) e 1:20 (2 aves).This is the first report of Toxoplasma gondii infection in black vultures (Coragyps atratus), which are obligate scavengers found throughout the Americas. Serum samples from 121 wild black vultures caught in urban areas of the city of Sao Paulo, Brazil, were tested for the presence of T. gondii antibodies using the modified agglutination test (MAT; cutoff point 1:5). T. gondii antibodies were found in 16 birds (13.2%), with titers of 1:5 (6 birds), 1:10 (8 birds), and 1:20 (2 birds)